Supercritical Fluid Extraction - ACS Symposium Series (ACS

Chapter 18

Supercritical Fluid Extraction Developing a Turnkey Method 1

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C. R. Knipe , D. R. Gere , and Mary Ellen P. McNally

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Hewlett-Packard Company, P.O. Box 900, Route 41 and Starr Road, Avondale, PA 19311-0900 Hewlett-Packard Company, 39550 Orchard Hill Place Drive, Novi, MI 48050 Agricultural Products, Experimental Station, E402/3328B, E. I. du Pont de Nemours and Company, Wilmington, DE 19880-0402

Downloaded by UNIV OF ARIZONA on January 18, 2013 | http://pubs.acs.org Publication Date: May 8, 1992 | doi: 10.1021/bk-1992-0488.ch018

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Supercritical fluid extraction (SFE) is a new and promising technique for sample preparation. Because S F E is so new, and there are so many control parameters available to the scientist, it can be difficult to know how to proceed when developing an S F E method. This paper discusses two samples and describes a systematic approach for evaluating S F E extraction parameters for methods development. The effects of modifiers are investigated.

Although supercritical extraction (SFE) has been known for some time, it is still a relatively new technique to the analytical chemist. Before developing an S F E method, the chemist must understand the composition of the matrix and the analyte properties. The key instrumental parameters affecting the extraction of analytes from the matrix include: fluid density, temperature, and fluid composition. Both the make-up of the matrix and the analytes must be considered when selecting the extraction conditions. Consideration of the extraction parameters must be given with respect to their affect on the analytes of interest and on the compounds present in the matrix that may either coextract with the analytes or inhibit their extraction by physical or chemical means. Choice of the extraction fluid density will be dictated by the volatility and polarity of the compounds to be extracted. In general low density C 0 will 2

extract volatile non-polar compounds while higher density C 0 will generally 2

extract less volatile more polar material. The useful extraction temperature range will be influenced by the vapor pressure and thermolability of the analytes. The extraction temperature can also alter the physical state of the matrix itself, for example, liquifying fats oils and low molecular weight polymers. This in turn will affect recoveries and extraction 0097-6156/92/048g-0251$06.00A) © 1992 American Chemical Society

In Supercritical Fluid Technology; Bright, F., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1992.


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times necessary to remove the analytes of interest. The temperature will also determine the amount of modifer that can be solubilized by the extraction fluid. The choice of extraction fluid along with selection of modifier and amount of modifier will also affect extraction recoveries and extraction selectivity. In this work the extraction fluid chosen was CO2 as well as CO2 with the addition of a variety of organic modifiers. In addition to these extraction parameters the matrix itself will have a large impact upon the choice of the extraction conditions. The matrix, besides containing coextractable materials will impact the solute-solid interaction kinetics and thermodynamics. The coextractants themselves can also act as modifiers and, as their concentrations change over the course of an extraction, the make-up of the extraction fluid will dynamically change. Distribution of solutes throughout the matrix can also affect the extraction process. The analytes can be adsorbed on the matrix surface or encapsulated in the matrix. Encapsulated analytes may require harsher extraction conditions or additional preparation steps such as grinding or chopping, prior to extraction. Once this information is collected the chemist can address the problem of developing the extraction method.

Developing a Method Extraction Parameters. The challenge in S F E is to determine the best extraction parameters for the analytes of interest in a particular matrix. These parameters are listed in Table I. The solvent power of the extraction fluid as controlled by the density, temperature, and composition is the primary parameter which must be controlled to obtain optimal recoveries. The definition of solvent power used here is that of Snyder and Kirkland (1). As previously mentioned low densities will extract non-polar volatile compounds and high densities will extract relatively more polar, less volatile compounds. A quick way to evaluate the density effect is to do several extractions at different densities (low, medium, and high) to see when the analytes begin to extract (2). Another parameter to control is the extraction temperature. In addition to affecting the density, raising the temperature can add thermal energy to the system. This can either increase the partial pressure of the solutes or reach the melting point of the matrix, thereby freeing up the solutes, and allowing them to be transported from the matrix by the extraction fluid. The increase in temperature will also increase the diffusivity of fluids above their critical temperature. Another parameter that can have a large effect on the extraction process is the addition of modifiers. The effects of modifiers are still not well understood, but they will change the solvent power of the fluid and/or change the solute/solid interaction (generally both). After the fluid characteristics have


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Developing a Turnkey Method

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Table I. Key Extraction Parameters


Physical parameter

Experimental parameter

Solvent power

Fluid density Temperature Fluid composition

Solvent contact

Flow rate Extraction time Extraction mode (static/dynamic)

been determined the fluid/sample interaction time and the fluid volume must be specified. This interaction is determined by flow rate, extraction time, and whether or not the extraction is done in a static or dynamic mode. Analyte Properties. Once the analytes of interest are extracted, they must be collected/trapped for analysis and quantitation. Once collected/trapped they may have to be reconstituted, filtered, or derivatized prior to final analysis. Again the chemical/physical properties of the analytes will determine the collection and reconstitution rinse parameters . During the extraction step, the volatility of the analytes will determine the collection temperature or type of adsorbent material used for collection. If the analytes are volatile, then a cold trap or cooled collection solvent along with a low flow rate should be used. This is because the analytes are volatile and the expansion of the C 0 can create aerosols or mechanically move the analytes past the collection device. Less volatile analytes can tolerate higher extraction flow rates and higher collection temperatures can be used. If an adsorbent trap is used for collection, the chemist can specifiy an appropriate adsorbent and rinse solvent for optimal recoveries for the analytes of interest^). Flow rate and volume are parameters that also need to be specified. The flow rate and rinse volume are determined by the solublity of the analytes in the rinse solvent and the amount of material to be removed from the trap. 2

Two examples were chosen to show the effect of these S F E parameters and the process of developing a method. The first example is the extraction of paprika and an examination of some of these extraction parameters in a qualitative way. The second example, developing a method for the extraction of several herbicides from soil, shows a more quantitative approach. The herbicide


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example demonstrates the significant effect that modifiers can have upon extraction recoveries. It also shows that the solute/matrix interactions are as important as the solute/solvent interactions when developing an S F E method.


Qualitative Method Paprika Analysis. Paprika was selected because it has a wide range of readily extractable compounds. There are essential oils, aroma components, and carotenoids contributing to color and taste. Paprika is readily available and provides various extracts easily detectable by sight and smell. Thus using paprika as a sample yields a large amount of qualitative information that can be quickly and easily gleaned concerning the effect of varying S F E extraction conditions. Fractionation of Paprika. Since there are so many extractable components in paprika, it helps to simplify the analysis by being somewhat selective during the extraction. The solvent power of the fluid will have the most impact on the selective extraction of certain classes of compounds from the sample. Selective extractions were obtained by extracting the sample multiple times at different densities, commonly called density stepping or fractionation (as discussed by W.S. Miles at the Pittsburgh Conference in New York City 1990, paper 543).

Table II. Paprika Extraction Conditions Experimental: Sample Size: 10-300 mg of store bought paprika. Chamber temperature 40 and 80 ° C . Extraction fluid: C 0 . 2

Flow rate: 4 ml/min mass flow of liquid C 0 at the pump. Thimble volumes: 10 Thimble size: 1.5 ml. Extraction time: 1.0, 2.43, 3.25, 3.85 min Density: 0.25, 0.6, 0.8, 0.95 g/ml. Extractor: HP7680A SFE. 2

In this case both density and temperature were varied to investigate qualitatively the conditions at which the various compounds would be extracted from the paprika. Table II indicates the extraction conditions used for the paprika sample.


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Step I. The first set of experiments involved extraction of paprika using pure C 0 at three different densities; 0.25, 0.6, 0.95 g/ml and at a temperature of 40 2

° C . As expected, the first collection vials came out clear, suggesting that if anything was extracted, it was limited to the more volatile non-polar aromatic components or oils. The second extraction step, at a density of 0.6 g/ml C O ^ produced a pale orange extract. This suggests that some of the less volatile more polar compounds were extracted. The third extraction step, at a density of 0.95 g/ml C 0 , produced a much darker orange color. A t this higher density of C 0 the carotenes and carotenoids were extracted. This confirmed the expectation that extraction of less volatile, more polar compounds occurs at the higher densities.


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Step II. Next, the same S F E extractions were rerun using the same flow rates and thimble volumes as for step I, but at a chamber temperature of 80 °C. The same densities were used except that 0.8 g/ml was substituted for 0.95 g/ml at the new temperature. The density of 0.8 g/ml at 80 °C was used because of the upper pressure limit of the extractor. The upper pressure limit of the extractor was 400 atm., therefore at 80 °C, this corresponds to a density of 0.80 g/ml for C 0 . In this case, the extracts collected at 0.6 and 0.8 g/ml densities were 2

darker in color than the corresponding extracts for those densities at 40 °C. This indicates that an elevated temperature can aid in the extraction of colored components (which are typically more polar, less volatile components) from the paprika with SFE. Without some quantitative results it is difficult to say what the differences are in relation to specific compounds. Nor does this finding supply specific information about how much additional material was extracted at 80 °C versus 40°C, all other conditions being equal. The results for step II are summarized in Table III. Table III. Extracted Paprika Fractions Extraction Density

Extract

Temperature = 40 °C. 0.25 g/ml 0.60 g/ml 0.95 g/ml

Colorless extract. Pale orange extract. Orange extract.

Temperature = 80 °C. 0.25 g/ml 0.60 g/ml 0.80 g/ml

Colorless extract. Orange extract. Dark orange extract.


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Thimble Volumes. To properly compare extractions while changing the density, the number of thimble volumes swept was kept constant for all extraction steps. In this case the extraction time was changed as a new density setpoint was chosen for each extraction step while the flow rate at the pump head was kept constant. The pump head is where the flow of liquid C 0 is controlled. Control of the liquid C 0 controls the mass flow of the system. As the density and the extraction times changed, so did the mass of C 0 used per step; however, the net volume of solvent seen by the sample did not change. Importantly, as the solvent changed from 0.25 g/ml C 0 to a solvent of 0.95 g/ml C 0 , more C 0 was required to displace the same volume element. Since the mass flow was held constant in this set of experiments the extraction time had to be changed to normalize for equivalent volumes of solvent as seen by the sample. 2

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Quantitative Method Herbicide Extraction. Traditionally one of the more time consuming and tedious sample preparation problems has been the extraction of herbicides and pesticides from soil. Due to Federal regulations these agrochemical products must be monitored, and the number of samples that need to be analyzed can be significant^. Along with the large number of samples that must be analyzed there is a need for improved sample preparation methods that minimize the preparation time and solvent usage, such as SFE. Two families of herbicides that require monitoring are the s-triazines and the phenylureas. The triazines selected for extraction were atrazine and cyanazine, and diuron was selected from the phenylureas(5)(6). Table IV shows some of the physical properties of these herbicides. A l l three have similar melting points and molecular weights. They are relatively small molecules and are not highly polar and so should be

Table IV. Herbicide Properties Atrazine Melting Point Molecular Weight Vapor Pressure

Cyanazine

Diuron

171-174 °C 167-169 °C 158-159 °C 215.68g/mol 240.68g/mol 233.10g/mol 0.113 mPa 0.21 //Pa 0.25 mPa


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amenable to extraction using supercritical C 0 . The vapor pressures show that they are not highly volatile, so a technique such as headspace analysis can be ruled out. 2


Spiked Celite. To determine extraction conditions, a standard solution of the three herbicides was extracted from celite. The celite provided a relatively inert solid matrix for the solutions to adsorb onto. Since the celite is inert, this eliminates many of the solute/solid interactions, thereby providing information about the extraction conditions that concern primarily just the solvent/solute interactions.

Table V. Herbicide Extraction Conditions Extraction Conditions

Density: Flow rate:

Step 1 0.25 1.0

Temperature: Fluid: Extraction time: Thimble volumes:

50 °C C0 9.4 minutes 5

Step 2 0.60 2.4

Step 3 0.9 g/ml 3.6 ml/min

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200/*l of a standard solution was pipetted onto approximately 1 gram of celite. The solvent was allowed to evaporate and the celite was placed into the extraction chamber. The spiked celite was then extracted at three densities; 0.25, 0.6, 0.9 g/ml C 0 . The extraction time for the steps was selected to allow five thimble volumes to be swept for each step. The full set of extraction conditions is shown in Table V . 2

Thimble Volumes. For these experiments the thimble volumes and the extraction times were held constant. To accomplish this, the mass flow of the system had to be varied by changing the flow rate at the pump head for each density step. Controlling the mass flow rate allowed the linear/volumetric flow to be consistent throughout the experiments. This is different from the paprika experiments in which the mass flow was held constant and the extraction times were changed, to keep thimble volumes constant for each extraction step. Flow control is one of the major advantages of variable restrictor based S F E units.


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The variable restrictor allows the user to control both the mass flow of the system and the density independently.

Table VI. Analytical Conditions


Chromatographic Conditions: Gas Chromatograph: H P 5890 Series II Column: Supelco SPB-5 column (530 //m x 15 m) Detector: Nitrogen phosphorus detector (NPD) Injector: Cool on-column Temperature program: Ramp 35 °C - 250 °C Injection volume: 2^1

Spiked Celite Recoveries. Recoveries were determined by analyzing the extracts by gas chromatography using a nitrogen phosphous detector(NPD). (See Table VI.). A calibration curve was generated using the standard solution that was used to spike the celite. To quantitate the extracts, the collection vials were diluted to volume and the G C / N P D area counts were compared to the calibration curves. Recovery results from this density stepping showed that the atrazine was soluble at all densities of C 0 , while cyanazine and diuron did not extract well until the density of the C 0 increased to 0.9 g/ml. The results are shown in Table VII. This indicates the need to use a high density to extract these solutes from soil. 2

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Table VII. Recovery from Celite Density

Diuron

Atrazine

Cyanazine

0.25 g/ml 0.60 g/ml 0.90 g/ml

32.5% 47.2% 23.3%

1.0% 5.3% 58.2%

1.1% 7.7% 60.8%

Total

103.0%

64.5%

69.6%

Spiked Soil Samples. To be sure that the soil matrix did not contribute extractable materials in addition to the analytes of interest, a soil blank was extracted at a density of 0.9 g/ml of C 0 as a control. Gas chromatography using 2


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flame ionization detection (FID), showed that very few co-extractants were present in the matrix. Using an element specific detector to analyze for these herbicides (NPD), can provide a false sense of security when considering coextractants and their impact upon the extraction method. The density stepping method was then run on a spiked soil sample. Five grams of soil were spiked with the same solution of herbicide standards used to spike the celite sample and the solvent was allowed to evaporate. The spiked sample was then placed into an extraction thimble and extracted. The results were different than those obtained from the spiked celite. Even at a high density of C 0 the herbicides were not extracted. Previous experience showed that using water as a modifier aided in the extraction of diuron from soils (6)(7). Therefore 1 ml. of water was added to the spiked soil and the sample rerun at a density of 0.9 g/ml of C 0 . The results showed a significant increase in recovery of the herbicides with the addition of water. This demonstrates that the addition of a modifier added to the extraction cell can have a significant effect upon the extraction recoveries (8) (9). The results are summarized in Table VIII.


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Table VIII. Recovery from Soil Density

Atrazine

Cyanazine

Diuron

0.25 g/ml 0.60 g/ml 0.90 g/ml

0% 1.2% 1.0%

0% 1.1% 1.6%

0% 0.5% 0.9%

Total recovery

2.2%

2.7%

1.4%

0.90 g/ml (1.0ml H 2 0 added)

32.5%

33.5%

28.4%

The results of extracting these herbicides from celite versus soil, display a marked difference (as seen by comparing Table VII to Table VIII). The only difference between the extraction from celite and the extraction from soil was the matrix itself, all other conditions were identical. This raises the question; is the water modifying the solvent or modifying the matrix?


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Modifier Effects As a result of the increased recovery when water was added as a modifier, it was decided to look at the effect of various modifiers upon the extraction recoveries in a systematic manner.


Table IX. Solute Solubilites Solvent

Atrazine

Cyanazine

Diuron

Water Methanol Ethanol Chloroform Hexane

70 ppm 18,000 ppm NA 52,000 ppm NA

171 mg/1 NA 45 g/1 210 g/1 15 g/1

42 ppm NA NA low low

N A : Solubility data was not available.

Table I X shows the solublity of the herbicides in various solvents. If the extraction process is based on a solvation mechanism then a non-polar solvent (such as chloroform) should extract these solutes better than a more polar solvent (such as methanol or water). Modifiers were selected for addition to the spiked soil samples representing a range of solvent polarities. Based on the results of the density stepping experiments, the extraction times were increased to 19 minutes from 9.4 minutes, which increased the thimble volumes from 5 to 10. To be consistent throughout the experiments, 1 ml of modifier was added directly to each extraction cell. Methylene chloride was chosen as a non-polar modifier. Results show that the methylene chloride did little to aid in the extraction process for any herbicide. Next, more polar modifiers were selected, isopropyl alcohol and methanol. Both alcohols helped increase the recoveries from the soil, with the methanol being slightly better than the isopropyl alcohol. These results indicate that the more polar the modifier, the better the extraction recoveries. It required a very polar modifier (water), to obtain recoveries from the spiked soil comparable to those obtained from the unmodified spiked celite samples. Tables X , X I and XII show the herbicide recoveries and the relative solublities of these herbicides in various solvents. The results are not what one would expect based upon a solvation model of extraction. Since the herbicides are more soluble in non-polar solvents (Table IX), one would expect that a non-polar extraction fluid, such as C 0 or C 0 2


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modified with a non-polar solvent, would produce the greatest recoveries. The results from the spiked soil samples show that the opposite occurs, polar modifiers increase recoveries much more than non-polar modifiers. This indicates that the solute/matrix interactions were the interactions being modified versus the solute/solvent interactions. These results are in opposition to the practice of enhancing recoveries by changing the solvent power of the extraction fluid, using density, temperature or fluid composition.


Table X. Recovery of Atrazine Modifier (1.0 ml added)

Soil

Celite

None

0%

96.8%

Methylene chloride 4.1%

117.4%

Isopropyl alcohol

27.2%

15.9%

Methanol

49.8%

45.8%

Water

108.9%

94.6%

Relative Solubility 52,000 ppm chloroform 18,000 ppm in methanol 70 ppm in water

Table XI. Recovery of Cyanazine Modifier (1.0 ml added)

Soil

Celite

None

0%

96.8%


116.6%

Isopropyl alcohol

33.0%

15.7%

Methanol

52.3%

47.5%

Water

106.4%

81.8%

Relative Solubility 210 g/1 in chloroform 45 g/1 in methanol 171 mg/1 in water


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Table XII. Recovery of Diuron Modifier (1.0 ml added)

Soil

Celite

None

0%

96.8%


108.7%

Isopropyl alcohol

16.8%

12.6%

Methanol

35.8%

43.1%

Water

91.7%

93.3%

Relative Solubility low, in hydrocarbon solvents 42 ppm in water

The same experiments with modifier were run on spiked celite. The results were similar to those seen with the soil samples except in the case when methylene chloride was added to the celite. With the methylene chloride added as the modifier, the herbicides were totally extracted as in the unmodified experiments. One possible explanation is that the methylene chloride itself was being removed from the matrix more readily than the other modifiers, along with the herbicides. The alcohols may have inhibited the full recovery of the herbicides under these conditions by providing a liquid phase which could solublize the herbicides. The herbicides could then partition themselves between the liquid alcohol phase and the C 0 / a l c o h o l phase. This would lead to the herbicides being retained on the celite until the all of the alcohol modifier was removed by the C 0 extraction fluid. 2

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Modifier Addition Using Premixed Cylinders. The use of pre-mixed modifiers and C 0 was also investigated. A 5% by weight mixture of isopropyl alcohol and C 0 , and a 7% by weight mixture of methylene chloride and C 0 , were used under the same extraction conditions. These mixtures represent approximately a 4 mole % mix of each modifier with C 0 . Under these extraction conditions this corresponds to 3.6 - 4.4 ml of liquid modifier being used per extraction instead of the 1.0 ml volume added directly to the extraction thimble. The recoveries 2

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obtained by using modified tanks were generally lower than those obtained by adding the modifier directly to the extraction thimble. (See table XIII). The premixed modifiers enter the extraction cell already in the CO2 fluid phase. The modifier stays in the fluid phase during the extraction, therefore has less interaction with the matrix itself compared to the liquid modifier being placed directly on the sample(70,). This reduced interaction with the matrix is probably responsible for the lower recoveries. A l l of these experiments were done in a dynamic mode, the results might have been quite different if the experiments with the modified tanks were run in a static mode. Under static conditions the modified C 0 would have a longer time to interact with the matrix and potentially produce higher recoveries.


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Table XIII. Different Methods of Modifier Addition Modifier

Atrazine

Cyanazine

Diuron

15.9%

15.7%

12.6%

6.5%

7.9%

7.5%

Celite Isopropyl alcohol

a

5% IPA in cylinder

b

Methylene chloride

a

7% M e C l in cylinder^ 2

117.4%

116.6%

108.7%

69.2%

70.0%

65.6%

27.2%

33.0%

16.8%

0%

0%

0%

4.1%

11.3%

4.0%

8.7%

18.8%

7.8%

Soil Isopropyl alcohol

a

5% I P A in cylinder

b

Methylene chloride

a

7% M e C l in cylinder** 2

a) 1.0 ml of modifier added directly to the sample b) (wt/wt)% for modifier in cylinder.

Reliability. The robustness of S F E as a routine technique is shown in Table X I V . These data were generated by loading a sample tray and the samples were run overnight with the help a robotic manipulator. The celite was spiked with the water in batch mode but extracted sequentially. It can be seen from the data that having the samples sitting at room temperatures had no deleterious effects upon


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the extraction recoveries. The relative standard deviation values include not only the extraction variations but all variations from sample preparation (spiking) and analysis.


Table XIV. Repeatability Data for Recoveries from Celite Matrix

Atrazine

Cyanazine

Diuron

Dry Dry 1.0 ml H 0 added 2

96.8% 96.0% 88.0%

97.2% 97.1% 82.0%

95.3% 91.3% 83.7%

1.0 ml H 0 added 2

94.6%

81.8%

93.3%

1.0 ml H 0 added

86.7%

86.8%

90.6%

Relative std. dev.

4.6%

7.7%

4.3%

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Summary S F E can be a fast and efficient method for sample preparation. The paprika sample demonstrates how techniques such as density stepping can rapidly give qualitative information concerning starting points for further S F E method development. However to properly develop an S F E method requires a systematic approach for varying the extraction parameters.. The herbicide method was developed by first extracting a standard solution from an inert matrix (celite). These results showed that supercritical C 0 could extract the herbicides of interest. However when these same S F E conditions were used to extract the herbicides from a spiked soil sample, the recoveries were quite different. Several modifiers were added to the spiked soils. The modifiers were selected to represent a wide range of polarity from non-polar (methylene chloride) to polar (water). These results showed that the modifiers producing the best recoveries, were those that the herbicides were least soluble in, suggesting that these modifiers are modifying the solute/matrix interaction not the solute/solvent interaction. Results also indicate that it is more efficient to apply the modifier directly to the matrix versus mixing the modifier with the C 0 , prior to introducing it into the extraction cell. 2

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Literature Cited 1. Snyder, L . R.; Kirkland, J.J., Introduction to Modern Liquid Chromatography; John Wiley & Sons, Inc.: New York, N Y , 1979; pp. 257-258.



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2. Giddings, J.C.; Myers, M . N . ; King, J.W.J. of Chrom. Sci. 1969, 7, pp. 276-283. 3. Mulcahey, L.J.; Hedrick, J.L.; Taylor, L.T. Anal. Chem. 1991, 63, pp.22225-2232. 4. Buser, H-R. Environ. Sci. Technol. 1990, 24, pp. 1049-1058. 5. Janda, V.; Steenbeke, G.; Sandra, P. J. of Chrom. 1989, 479, pp. 200-205. 6. Hance, R.J. Weed Res. 1965, 5, pp. 108-114. 7. McNally, M . E . ; Wheeler, J.R.J. of Chrom. 1988, 447, pp. 53-63. 8. Dooley, K.M.; Ghonasgi, D.; Knopf, F.C. Environ. Prog. 1990, 9, pp. 197-203. 9. Wheeler, J.R.; McNally, M . E . J . of Chrom. Sci. 1989, 27, pp. 534539. 10. Berger, T.A.; HRC 1991, 14, pp. 312-316. R E C E I V E D December 2, 1991


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